Listeria monocytogenes incubation temperature

Equipment and materials

FDA does not specifically endorse any of commercial products listed. Equivalent products may be available.

1. Sterile swab: the following or equivalent:
   • 3M™ Swab-sampler in 10 ml D/E neutralizing broth [Catalog# RS96010DE, www.mmm.com] [Polyester].
   • Puritan dry cotton swab [Catalog# 25-806 1PC, 25-806 2PC, www.puritanmedproducts.com] [Cotton]
   • World Bioproducts PUR-Blue™ swab sampler with [Catalog #BLU-10DE] or without  [Catalog# BLU-DRY, www.worldbioproducts.com] 10ml D/E neutralizing broth [Polyurethane].
   • Healthlink® dry swab transporter [Catalog# 4159BX, www.hardydiagnostics.com] [Polyester].
2. Sterile sponge: the following or equivalent:
   • World Bioproducts EZ Reach™ sponge sampler with [Catalog# EZ-10DE-PUR] or without [Catalog# EZ-DRY-PUR, www.worldbioproducts.com] 10 ml D/E neutralizing broth [Polyurethane].
   • Nasco Whirl-Pak® dry sponge probe [Catalog# B01475WA, www.enasco.com] [Cellulose].
   • 3M™ Sponge-sticks with [Catalog# SSL10DE, www.mmm.com] or without [Catalog# SSL100] 10 ml D/E neutralizing broth [Cellulose].

   If available, dry samplers can be obtained and D/E neutralizing broth added later, or pre-moistened samplers can be obtained. We do not recommend less than 10 ml D/E broth due to the possible presence of sanitizer residues on environmental surfaces.

3. Balance for weighing sample to 0.1 g
4. Incubators, 30 and 35°C
5. Water bath, 80°C ± 2°C
6. Phase-contrast microscope with oil immersion phase objective [100×]
7. Blender motor and jars or Stomacher and bags
8. Vortex mixer

Media and Reagents

FDA does not specifically endorse any of commercial products listed. Equivalent products may be available.

1. Buffered Listeria Enrichment Broth [BLEB] [M52]
2. Acriflavine monohydrochloride
3. Nalidixic acid [sodium salt]
4. Cycloheximide
5. Natamycin [Pimaricin]
6. Dey/Engley [D/E] broth [M193]
7. Oxford medium [OXA] [M118]
8. PALCAM agar [M118a]
9. MOX agar [M103a]
10. Lithium chloride-phenylethanol-moxalactam [LPM] agar [M81] with added esculin and iron [M82]
11. R&F Listeria monocytogenes Chromogenic Plating Medium [R&F Laboratories, Downers Grove, IL] [M17a]
12. ALOA agar [M10a]
13. Chromogenic Listeria Agar [Oxoid Ltd, Basingstoke, England] [M40b]
14. Rapid’ L.mono [BioRad Laboratories Inc.] [M131a]
15. CHROMagar Listeria [CHROMAgar, Paris, France] [M40a]
16. Trypticase soy agar with 0.6% yeast extract [TSAYE] [M153]
17. Sheep Blood Agar [M135]
18. Hydrogen peroxide solution, 3% for catalase test [R12]
19. Gram stain kit
20. Motility test medium [MTM, Difco] [M103]
21. Trypticase soy broth with 0.6% yeast extract [TSBye] [M157]
22. Purple carbohydrate fermentation broth base [M130], containing 0.5% solutions of dextrose, esculin, maltose, rhamnose, mannitol, and xylose
23. Physiological saline solution, 0.85% [R63]
24. Fluorescent antibody [FA] buffer [Difco]
25. Listeria-typing sera Type 1 [Difco cat. # 223031] and Type 4 [Difco cat. # 223041]
26. Listeria Antisera Set [Denka Seiken product #294616]
27. Optional: Nitrate reduction medium [M108] and nitrate detection reagents [R48]

Note: Alternative companies may be used when the products are equivalent.

Control Cultures

1. Listeria monocytogenes ATCC 19115
2. Listeria innocua ATCC 33090
3. Listeria seeligeri ATCC 35967
4. Listeria ivanovii ATCC 19119
5. Rhodococcus equi ATCC 6930
6. Staphylococcus aureus ATCC 25923 or ATCC 49444

Sample Preparation

Sample transport and storage practices should maintain the recommended storage conditions for the food commodity. Sample analysis should be initiated as soon as possible upon sample receipt. If sample analysis must be delayed, store frozen samples frozen [-20 °C ± 5 °C]; store nonperishable, canned or low-moisture foods at room temperature, and store refrigerated, unfrozen perishable foods at 4 °C ± 2 °C until sample analysis is initiated.

Basic analytical options include: 1. Qualitative detection [limit of detection

1. Qualitative detection from foods and environmental samples 
   1. Individual subsample analysis: For solids, semi-solids, or liquids add 25g representative portion to 225 ml BLEB containing pyruvate without selective additives [M52]. [10, 26]. Thoroughly mix, blend or stomach, continue enrichment as described in section E or F. Certain foods may require different sample set-up procedures such as soaking and rinsing. A 50 g portion of the sample should be reserved for possible pathogen enumeration. Store it at 5° C if it is not frozen or, if frozen, in a non-defrosting freezer. Refer to applicable sampling compliance guidance documents for additional instructions.
   2. Composite sample analysis: composites may be used to analyze multiple sub units from a single sample. Refer to applicable sampling compliance guidance documents for specific composite procedures. Generally, two composites are prepared from a sample consisting of 10 sub-samples [liquid, cream or solid food]. To prepare a composite, 50-g or ml representative portions from each of the 5 sub-samples are pooled and 250 ml basal BLEB without selective agents is added and then blended or stomached [M52]. A 50 g portion of this composite blend [equivalent to 25g food plus 25 ml basal BLEB] is combined with 200 ml of basal BLEB. The composite is then inoculated as described in section E or F. An aliquot [100 ml] of the composite blend should be retained, preferably at 5° C and not below 0° C, for possible pathogen enumeration.
   3. Environmental samples: When sampling dry surfaces, swabs [cotton, polyester or polyurethane] or sponges [cellulose or polyurethane] should be pre-moistened in 10 ml Dey/Engley [D/E] neutralizing broth. Swabs are typically stored in tube containers and sponges are typically stored in bag containers. Before sampling, press swabs against inner tube walls or squeeze the sponges in bags to remove excess broth. Swab environmental surfaces using firm and even pressure vertically [approximately 10 times], then flip the sampler and use the other side to swab horizontally [approximately 10 times] and diagonally [approximately 10 times]. When putting the samplers back to the container, make sure they are submerged in the D/E neutralizing broth. When sampling wet surfaces, dry swabs or sponges should be used and put into D/E neutralizing broth immediately after sampling.  Swabs that are made from cotton or that have large tips appear to absorb liquid better than others. For samples that are not suitable to be collected by regular sized swabs/sponges mentioned in section A, refer to applicable sampling compliance guidance documents for additional instructions. After sampling, swab/sponge can be maintained in D/E broth at 4°C for up to 48 h before analysis. Swab/sponge and D/E neutralizing broth are then added to 90 ml [or more to fully submerge the swab or sponge] of BLEB containing pyruvate without selective additives [M52]. Sponge and D/E broth can also be added to 225ml of BLEB. Thoroughly massage or stomach to expel the collection broth into enrichment broth. Continue enrichment as described in section E or F.
   4. Interpret result. Confirmation of one or more L. monocytogenes isolates from an enrichment indicates that L. monocytogenes is present at ≥ 1 CFU per sample size analyzed or present on environmental swab or sponge sample.
2. Quantitative determination from foods. Enumeration is performed using a combination of MPN and direct plating. Refer to section J for details. Surveillance samples are generally first analyzed by qualitative detection and reserve portions of the positive samples are then enumerated. For outbreak response situations, all samples may be directly enumerated. Refer to section J and applicable sampling compliance guidance documents.

The following alternative screening methodologies may be used to screen samples for the presence of Listeria. Follow the manufacturers' package insert making certain they have not deviated from the approved versions of the AOAC INTERNATIONAL Official Methods Manual protocols [Section F1]. The kits are only approved for the specified food matrices, claimed in the OMA method, which vary from kit to kit. For other food matrices that are not validated a matrix extension validation is necessary. Negative results obtained with the products are considered definitive and no further testing is required. Presumptive positive results with these rapid screening methods must be confirmed by streaking to the selective agars and confirming isolates to the species level by the procedures described in sections G-I.

F1. Rapid screening methods for Listeria spp.

F2. Rapid screening methods for Listeria monocytogenes. Please note that these methods do not screen for Listeria spp. and therefore may not be suitable for situations in which the identification of Listeria spp. is desired.

1. 1. AOAC Official Method 993.09. Listeria in select foods Colorimetric deoxyribonucleic acid hybridization method [GENE-TRAK Listeria Assay]. [3, 16]

      [Applicable to dairy products, meats, and seafoods]

      Collaborative study: milk [2%], brie cheese, cooked crab meat, frankfurters, roast beef, raw ground pork

      Pre-collaborative: crab meat, raw shrimp, cheddar cheese, cottage cheese, ice cream, chocolate milk, nonfat dried milk, fish fillet, ground raw pork, fermented sausage, raw ground turkey.

   2. AOAC Official Method 994.03. Listeria in select foods. Colorimetric Monoclonal Enzyme-Linked Immunosorbent Assay Method [Listeria-Tek] [4, 17, 31]

      [Applicable to dairy products, seafoods, meats]

      Collaborative study: frankfurters, roast beef vacuum packed, brie cheese, 2% milk, raw frozen shelled shrimp, cooked frozen crab.

      Pre-collaborative: crab meat, ice cream, milk, chocolate milk, non-fat dried milk, raw fish, cooked beef, roast beef, cured ham, raw sausage, raw oyster, raw chicken, raw turkey.

   3. AOAC Official Method 995.22. 2000. Listeria in select foods. Colorimetric polyclonal enzyme immunoassay screening method [TECRA Listeria Visual Immunoassay]. [5, 29]

      [Applicable to dairy foods, seafoods, poultry, meats [not raw ground chuck], leafy vegetables]

      Collaborative study: fish fillets, ice cream, lettuce, chicken, ground turkey

      Pre-collaborative: crabmeat, shrimp, soft cheese, chocolate milk, non-fat dried milk, raw beef, roast beef, frankfurters, bologna, oysters, chicken.

   4. AOAC Official Method 2002.09. Listeria in select foods. TECRA Listeria Visual

      Immunoassay Using TECRA Listeria Enrichment Broth. [32, 5, 29].

      [Applicable to raw and processed meats, cultured and non-cultured dairy products];

      Note: Method is based upon 995.22 but with altered enrichment, omission of cyclohexamide and additional foods.

      Collaborative study: fish fillets, turkey, raw ground beef, ice cream, lettuce.

   5. AOAC Official Method 996.14. Listeria in select foods Assurance Polyclonal Enzyme Immunoassay Method [EIA] [6, 19].

      [Applicable to dairy foods, red meats, pork, poultry products, fruits, nutmeats, seafood, pasta, vegetables, cheese, animal meal, chocolate, and eggs, bone meal and from environmental surfaces]

      Collaborative study: nonfat dried milk, ice cream, raw poultry, raw shrimp, cooked roast beef, green beans

      Pre-collaborative: crabmeat, soft cheese, dry egg, egg liquid frozen, milk, chocolate milk, raw fish, bone meal, raw beef, raw pork, scallops, chocolate, nuts, pasta, raw chicken, coleslaw

   6. AOAC Official Method 997.03. Listeria in select foods Visual Immunoprecipitate Assay [VIP] [7, 20].

      [Applicable to dairy foods, red meats, pork, poultry and poultry products, seafood, fruits, vegetables, nutmeats, pasta, chocolate,eggs, and bone meal, and environmental surfaces.

      Collaborative study: nonfat dried milk. Ice cream, raw poultry, raw shrimp, cooked roast beef, green beans, environmental surfaces

   7. AOAC Official Method 999.06. Listeria in select foods. Enzyme Linked Immunofluorescent Assay [ELFA] VIDAS LIS Assay Screening Method [8, 21].

      [Applicable to dairy products, vegetables, seafoods, raw meats and poultry, and processed meats and poultry]

      Collaborative study: ice cream, green beans, fish, turkey, cheese, roast beef

   8. AOAC Official Method 2004.06. Listeria in select foods. Modified VIDAS LIS Assay Screening Method [36].

      [Applicable to the detection of Listeria in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry.]

      Collaborative study: brie cheese, ice cream, fish, green beans, roast beef

   9. AOAC Official Method 2010.02. Listeria in select foods. VIDAS LSX Assay Screening Method [35].

      [Applicable to dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry.]

      Collaborative study: vanilla ice cream, cheddar cheese, raw ground beef, frozen green beans, deli turkey, cooked shrimp

   10. AOAC Official Method 2013.10. Listeria in select foods and environmental surfaces. VIDAS UP Listeria [LPT] Assay Kit [36].

       [Applicable to deli ham [25 and 125 g], pepperoni [25 g], beef hot dogs [25 g], chicken nuggets [25 g], chicken liver pâté [25 g], ground beef [125 g], deli turkey [125 g], cooked shrimp [25 g], smoked salmon [25 g], whole cantaloupe melon, bagged mixed salad [25 g], peanut butter [25 g], black pepper [25 g], vanilla ice cream [25 g], queso fresco [25 and 125 g], stainless steel, plastic, ceramic and concrete environmental surfaces.]

       See supplemental data, Tables 2A–D, for detailed results of the collaborative study on J. AOAC Int. website, //aoac.publisher.ingentaconnect.com/content/aoac/jaoac.

   1. AOAC Official Method 2003.12. Listeria monocytogenes in select foods. BAX® Automated System [9].

      [Applicable to dairy products, fruits and vegetables [except radishes], seafoods, raw and processed meats, and poultry.]

      Collaborative study: frankfurters, soft cheese, smoked salmon, ground beef, radishes, peas

   2. AOAC Official Method 2004.02. Listeria monocytogenes in select foods. Enzyme Linked Immunofluorescent Assay [ELFA] VIDAS LMO2 Assay Screening Method [33].

      [Applicable to the detection of L. monocytogenes in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry.] Collaborative study: vanilla ice cream, brie cheese, coked roast beef, frozen green beans, frozen tilapia fish

   3. AOAC Official Method 2013.11. Listeria monocytogenes in select foods. VIDAS Listeria monocytogenes [LMX] Assay Kit [37].

      [Applicable to deli ham [25 and 125 g], fermented sausage [25 g], liver pâté [25 g], processed cheese [25 g], vanilla ice cream [25 g], cooked shrimp [25 g], smoked white fish [25 g], frozen spinach [25 g], peanut butter [25 g], deli turkey [25 and 125 g], queso fresco [125 g], and ground beef [125 g].]